Mycoplasma pneumoniae is a pathogen of the human respiratory tract and the leading cause of pneumoniae in the age group that includes older children and young adults. The ability to adhere to the respiratory epithelium is an important virulence determinant and appears to be a complex process requiring the proper interaction of several specific groups of mycoplasma proteins. Despite the importance of cytadherence to colonization, M. pneumoniae undergoes phase variation in the cytadherence phenotype. The long term objective of this project is to elucidate through genetic means both the mechanism(s) regulating the coordinate expression of phase-variable cytadherence-associated proteins HMW1-4, as well as the specific roles of these proteins in the adherence event. The gene for HMW3 has been cloned and partially characterized, and preliminary evidence suggests that the gene for HMW1 has likewise been identified. The gene for HMW2 will be identified, and the identities of the candidate genes for HMW1-3 will be confirmed. Phase variable expression of HMW1-3 will be investigated at the level of transcription, and the nucleotide sequences of potential regulatory regions for these genes will be compared in wildtype and cytadherence- deficient variants. A physical map of the M. pneumoniae chromosome under construction will be completed and the relative positions of the HMW loci established. The clinical relevance of cytadherence phase variation will be evaluated by screening fresh clinical isolates and characterizing cytadherence-deficient variants isolated. Finally, developmental projects will be continued to enhance the expression of cloned mollicute DNA in Escherichia coli and to allow introduction of cloned genes back into M. pneumoniae.